During cold acclimation the heat production is shifted from shivering to non-shivering thermogenesis, which is mediated by adrenergic signaling. It has also been observed, that cold acclimation may increase the organismal resistence to pathological stimuli and may affect functional parameters of cardiovascular system. However, acute exposure to sever cold is often associated with detrimental effects on the body. We have recently shown that chronic exposure to cold increases the heart's resistance to ischemia-reperfusion injury without negative side effects when mild temperatures are used, however the mechanism of protection is not yet known. The aim of this work was to determine whether: i) if the sensitivity of the heart to ischemia changes already after the first day of cold exposure and does not show any negative effects, ii) if β1-adrenergic signaling plays a role in chronic regimen of cold-induced cardioprotection. The results of this work showed that i) one day of exposure to mild cold did not change the sensitivity of the heart to ischemia and ii) metoprolol treatment reduced the infarct size in the control group, but did not affect the heart of cold-adapted rats. Key words : Heart, rat, beta 1 adrenergic receptors, cold acclimation

Maintaining of homeostasis is essential for the survival of the organism. Stress disturbs the homeostasis and prepares the organism for mental or physical stress. During the stress situation, the endogenous stress factors are released. Through these factors stress affects tissue regeneration, the immune system and other metabolic processes. Chronic stress impacts many parts of body and mind and has a negative effect on these processes. Acute stress has the opposite effect. Mesenchymal stem cells (MSCs) participate in regenerative processes and modulate the immune system. Therefore, it can be assumed that stress will affect on MSCs. The aim of this study was to investigate the effect of stress factors, norepinephrine and corticosterone on the properties and function of MSCs in acute and chronic stress model. In our study, stress factors did not affect the morphology, vitality and differentiation of MSCs. However, the metabolic activity of MSCs was reduced regardless of the duration of their action. The action of stress factors also affected the production of some immunologically relevant molecules and proteins. Unfortunately, the results did not show a clear effect of stress factors on the lymphocyte modulation by MSCs. Key words: mesenchymal stem cells, catecholamines, adrenergic receptors,...

Cardiovascular disease (CVD) is the leading cause of death in the United States. A common feature in most cardiac pathologies is the dysregulation of beta-adrenergic receptors (β-AR) and changes in the extracellular matrix (ECM). The ECM maintains strength and normal organization of cardiac tissue, while fibrosis (connective tissue scarring) is necessary for repair of damaged cardiac tissue. However, the dysregulation of the ECM leads to a number of cardiac disease pathologies. Osteopontin (OPN) is a protein with diverse biological functions in regulating the ECM such as bone resorption and calcification, wound healing, cell adhesion, cell survival, and apoptosis. OPN is expressed at low levels in the heart but increases with injury by promoting collagen synthesis, cardiac fibroblast growth, and adhesions to ECM proteins. Furthermore, as the heart ages, increases in ECM reorganization leads to cardiac damage and failure. Several studies have examined the role of OPN in the heart, but to date, no studies exist on the role of OPN in response to β-AR signaling and cardiac remodeling or the role that aging plays in this response. The goal of this study was to examine the effects of OPN on cardiac ECM remodeling following chronic beta-adrenergic stimulation. We proposed that OPN expression increases cardiac remodeling and dysfunction following ISO treatment in the aging heart evidenced by increased fibrosis. For this study, young (4 months) and middle age (14 months) mice with (WT) and without (KO) the OPN gene were treated with isoproterenol (ISO) for 28 days. Echocardiography was used to assess cardiac function. Mice were euthanized, and the hearts were analyzed for fibrosis using Masson’s Trichrome Staining. Results showed ISO increased fibrosis in the WT-ISO, but not KO-ISO compared to the respective controls (SHAM, no ISO treatment) for the middle age mice (p≤0.05). Furthermore, the aged WT-ISO group exhibited a 3-fold increase in fibrosis compared to the young WT-ISO group. Results from echocardiography will be analyzed and we expect to see compromised cardiac function in the WT-ISO groups compared to KO-ISO groups. OPN is currently being examined as a potential biomarker in heart failure. The results from this study will provide new insight on changes in the cardiac vasculature in the aging heart following injury and the role OPN plays in this process.

Intravenous injection of 3-33 nmol/kg of substance P (SP) caused pressor and tachycardic responses in anesthetized rats. The responses were not blocked by a ganglion nicotinic receptor antagonist or by pithing. Pretreatment with reserpine blocked both responses. β-Adrenoceptor blockade attenuated only the tachycardic response, and α-adrenoceptor blockade attenuated only the pressor response. These findings indicated that the effects of SP to increase blood pressure and heart rate are due to sympathetic ganglion stimulation. Studies with adrenalectomized rats showed that stimulation of the adrenals by SP contributes to both responses but makes a greater contribution to the tachycardic response. These observations raise the possibility that the tachykinin innervation of sympathetic ganglia and the adrenal medulla may be involved in the local regulation of blood pressure and heart rate.

In situ hybridization is used for detection of RNA expression when conservation of tissue architecture is important. Most in situ hybridization protocols are written for tissues from animals (i.e., rat) which can be harvested and preserved rapidly. In contrast, human tissue is more difficult to obtain, hence in situ hybridization experiments must frequently be performed with less than optimal tissue preservation. This procedure details hybridization of a radiolabeled single-stranded RNA probe (riboprobe) to complementary sequences of cellular RNA in human tissue sections. This method enables detection of rare mRNA species in specific cell types of human tissue, offering distinct advantages over other in situ methods due to increased sensitivity. In particular, we have found that UV cross-linking and ribonuclease treatment protocols need to be altered for human tissues to ensure successful results, making this protocol unique to those previously described. In situ hybridization experiments can be performed using either DNA or RNA probes. RNA probes are advantageous since they form stable hybrids, are single-stranded, have little or no reannealing during hybridization, and can be synthesized to high specific activity. RNA probes can be readily created utilizing SP6, T3, or T7 promoters in both sense and antisense orientations to provide non-specific (control) and specific probes. Disadvantages of RNA riboprobes include a tendency for RNA to stick non- selectively more than DNA, and degradation by RNase (hence strict adherence to RNase-free precautions is mandatory during most of the protocol). The following protocol includes: (1) preparation of human tissues (tissue fixation and sectioning are highlighted as critical for probe penetration, preservation of tissue architecture, retention of tissue RNA, and overall success); (2) generation of radiolabeled riboprobes (total incorporation of radionucleotide is important to increase sensitivity; 35S was chosen as a compromise between excellent sensitivity, cellular resolution, and required exposure times (compared with 32p or 3H); non-isotopic methods have not been tested in a side-by-side comparison with 35S in human tissues by us, but theoretically might offer faster exposure times while maintaining high resolution); (3) hybridization conditions (stringency, temperature, washes, tissue dehydration); and (4) sample visualization (application of photographic emulsion, developing, fixing, staining, and counterstaining of individual slides).

Background - This study tested the hypothesis that β1-adrenoreceptor blockade modulates the angiotensin II (Ang II)-evoked neural release of norepinephrine (NE) and epinephrine (Epi) into the cardiac interstitial fluid (ISF) space in experimentally induced mitral regurgitation (MR) in the dog. Methods and Results - Normal dogs (n=8) were compared with dogs with MR of 2 (n=8) and 4 (n=6) weeks' duration and with dogs with MR treated with β1-receptor blockade (RB; extended-release metoprolol succinate, 100 mg QD; MR+β1-RB) that was started 24 hours after MR induction for 2 (n=6) and 4 weeks (n=8). Left ventricular end-diastolic dimension increased 20% as plasma Ang II levels increased >5-fold in both MR and MR+β1-RB dogs at 2 and 4 weeks. Ang II infusion into the left atrium produced increases in ISF NE and Epi in normal dogs, which were further increased in 2- and 4-week MR dogs but were restored to normal in 4-week MR+β1-RB dogs. Ang II infusion produced 4-fold increases in circulating NE and Epi in 2- and 4-week MR dogs that returned to normal in 4-week+β1-RB dogs. Left ventricular angiotensin-converting enzyme activity and ISF Ang II were increased in 4-week MR dogs but were decreased in 4-week MR+β1-RB dogs. Conclusions - β1-RB decreases renin-angiotensin system sympathostimulation and activation by attenuating the Ang II-mediated NE and Epi release into the cardiac ISF and circulation and by decreasing left ventricular angiotensin-converting enzyme expression in the early phases of volume overload.

Stimulation of β-adrenergic receptors (βARs) causes apoptosis in adult rat ventricular myocytes (ARVMs). The role of reactive oxygen species (ROS) in mediating βAR-stimulated apoptosis is not known. Stimulation of βARs with norepinephrine (10 μmol/L) in the presence of prazosin (100 nmol/L) for 24 hours increased the number of apoptotic myocytes as determined by TUNEL staining by 3.6-fold. The superoxide dismutase/catalase mimetics Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP; 10 μmol/L) and Euk-134 decreased βAR-stimulated apoptosis by 89±6% and 76±10%, respectively. Infection with an adenovirus expressing catalase decreased βAR-stimulated apoptosis by 82±15%. The mitochondrial permeability transition pore inhibitor bongkrekic acid (50 μmol/L) decreased βAR-stimulated apoptosis by 76±8%, and the caspase inhibitor zVAD-fmk (25 μmol/L) decreased βAR-stimulated apoptosis by 62±11%. βAR-stimulated cytochrome c release was inhibited by MnTMPyP. βAR stimulation caused c-Jun NH2-terminal kinase (JNK) activation, which was abolished by MnTMPyP. Transfection with an adenovirus expressing dominant-negative JNK inhibited βAR-stimulated apoptosis by 81±12%, and the JNK inhibitor SP600125 inhibited both βAR-stimulated apoptosis and cytochrome c release. Thus, βAR-stimulated apoptosis in ARVMs involves ROS/JNK-dependent activation of the mitochondrial death pathway.

To evaluate whether cervical spinal neurons can influence cardiac indices and myocyte viability in the acutely ischemic heart, the hearts of anesthetized rabbits subjected to 30. min of LAD coronary arterial occlusion (CAO) were studied 3. h after reperfusion. Control animals were compared to those exposed to pre-emptive high cervical cord stimulation (SCS; the dorsal aspect of the C1-C2 spinal cord was stimulated electrically at 50. Hz; 0.2. ms; 90% of motor threshold, starting 15. min prior to and continuing throughout CAO). Four groups of animals were so tested: 1) neuroaxis intact; 2) prior cervical vagotomy; 3) prior transection of the dorsal spinal columns at C6; and 4) following pharmacological treatment [muscarinic (atropine) or adrenergic (atenolol, prazosin or yohimbine) receptor blockade]. Infarct size (IS) was measured by tetrazolium, expressed as percentage of risk zone. C1-C2 SCS reduced acute ischemia induced IS by 43%, without changing the incidence of sudden cardiac death (SCD). While SCS-induced reduction in IS was unaffected by vagotomy, it was no longer evident following transection of C6 dorsal columns or atropinization. Beta-adrenoceptor blockade eliminated ischemia induced SCD, while alpha-receptor blockade doubled its incidence. During SCS, myocardial ischemia induced SCD was eliminated following vagotomy while remaining unaffected by atropinization. These data indicate that, in contrast to thoracic spinal neurons, i) cranial cervical spinal neurons affect both adrenergic and cholinergic motor outflows to the heart such that ii) their activation modifies ventricular infarct size and lethal arrhythmogenesis.

The intrinsic cardiac plexus represents a major peripheral integration site for neuronal, hormonal, and locally produced neuromodulators controlling efferent neuronal output to the heart. This study examined the interdependence of norepinephrine, muscarinic agonists, and ANG II, to modulate intrinsic cardiac neuronal activity. Intracellular voltage recordings from whole-mount preparations of the guinea pig cardiac plexus were used to determine changes in active and passive electrical properties of individual intrinsic cardiac neurons. Application of either adrenergic or muscarinic agonists induced changes in neuronal resting membrane potentials, decreased afterhyperpolarization duration of single action potentials, and increased neuronal excitability. Adrenergic responses were inhibited by removal of extracellular calcium ions, while muscarinic responses were inhibited by application of TEA. The adrenergic responses were heterogeneous, responding to a variety of receptor-specific agonists (phenylephrine, clonidine, dobutamine, and terbutaline), although α-receptor agonists produced the most frequent responses. Application of ANG II alone produced a significant increase in excitability, while application of ANG II in combination with either adrenergic or muscarinic agonists produced a much larger potentiation of excitability. The ANG IIinduced modulation of firing was blocked by the angiotensin type 2 (AT 2) receptor inhibitor PD 123319 and was mimicked by the AT 2 receptor agonist CGP-42112A. AT 1 receptor blockade with telmasartin did not alter neuronal responses to ANG II. These data demonstrate that ANG II potentiates both muscarinically and adrenergically mediated activation of intrinsic cardiac neurons, doing so primarily via AT 2 receptor-dependent mechanisms. These neurohumoral interactions may be fundamental to regulation of neuronal excitability within the intrinsic cardiac nervous system.

Sympathetic nerve activity increases in the heart during cardiac failure. Here, we hypothesized that β1 integrins play a protective role in chronic β-adrenergic receptor-stimulated cardiac myocyte apoptosis and heart failure. l-isoproterenol (iso; 400 μg/kg per hour) was infused in a group of wild-type (WT) and β1 integrin heterozygous knockout (hKO) mice. Left ventricular structural and functional remodeling was studied at 7 and 28 days of iso-infusion. Western blot analysis demonstrated reduced β1 integrin levels in the myocardium of hKO-sham. Iso-infusion increased heart weight:body weight ratios in both groups. However, the increase was significantly higher in WT-iso. M-mode echocardiography indicated increased left ventricular end-diastolic diameter, percentage of fractional shortening, and ejection fraction in the WT-iso group. The percentage of fractional shortening and ejection fraction were significantly lower in hKO-iso versus hKO-sham and WT-iso. Peak left ventricular developed pressure and left ventricular end-diastolic pressure measured using Langendorff-perfusion analyses were significantly higher in the WT-iso group (P<0.05 versus WT-sham and hKO-Iso). The number of TUNEL-positive myocytes was significantly higher in hKO-iso hearts 7 and 28 days after iso-infusion. The increase in myocyte cross-sectional area and fibrosis was higher in the WT-iso group. Matrix metalloproteinase-9 protein levels were significantly higher in WT-iso, whereas matrix metalloproteinase-2 levels were increased in hKO-iso hearts. Iso-infusion increased phosphorylation of c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 in both groups. The increase in c-Jun N-terminal kinase phosphorylation was significantly higher in hKO-iso (P<0.001 versus WT-iso). Thus, β1 integrins play a crucial role in β-adrenergic receptor-stimulated myocardial remodeling with effects on cardiac myocyte hypertrophy, apoptosis, and left ventricular function.